NEW DEVELOPMENT FOR PASTA-PRODUCTS: IMMATURE WHEAT AND FUNCTIONAL COMPONENTS

1. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 NEW DEVELOPMENT FOR PASTA-PRODUCTS: IMMATURE WHEAT AND FUNCTIONAL COMPONENTS M. G. D’EGIDIO*, M. A. PAGANI**, M. C. CASIRAGHI**, M. ZARDI** and C. CECCHINI* ** Università degli Studi Milano * CRA – Istituto Sper. Cerealicoltura Roma

2. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 NO ADVERSE EFFECTS in the past FOOD PROPERTIES • WELL-BEING • BETTER HEALTH • REDUCTION OF THE RISK OF DISEASES nowadays “A food can be regarded as functional if it is satisfactorily demonstrated to affect one or more target functions in the body beyond adequate nutritional effects, in a way that is relevant either to improve the state of health and well-being and/or the reduction of risk of disease” (ILSI, 2002) FUNCTIONAL FOOD D’ Egidio et al. New development for pasta products:immature wheat and functional components

3. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 n PREBIOTIC EFFECT FUNCTIONAL COMPONENTS FOS FRUCTOLIGOSACCHARIDES carbohydrates forming a short linear chain of fructose (2-9 units) with a single glucose unit at the head present as a plant storage carbohydrates in a number of vegetables and plants including wheat and other cereals, onion, bananas, garlic and chicory soluble in water non digestible in the small intestine (1.5 kcal/g) typically non viscous highly fermentable Mitsuoka et al.,1987; Tashiro et al., 1992; Spiegel et al., 1994 D’ Egidio et al. New development for pasta products:immature wheat and functional components

4. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 Studies have shown that a large quantity ofFOSis stored in the stems and grains of WHEAT for the first period of its growing cycle. 21 DAA 35 DAA 28 DAA 17 DAA 13 DAA 9 DAA MILKY PHASE WAXY PHASE PHISIOLOGICAL PHASE days after anthesis (DAA) IMMATURE WHEAT at “MILKY PHASE” moisture durum wheat is richer in FOS than common wheat D’ Egidio et al. New development for pasta products:immature wheat and functional components

5. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 The whole process is to be optimized for industrial scale IMMATURE WHEAT GRAINS (IWG) need a drying process in order to reduce their moisture content and so to be used as raw material in conventional processes LABORATORY SCALE EARS at MILKY PHASE MOISTURE  65% drying at 35°C 24- 48 h THRESHING MOISTURE KERNELS 13-15% MILLING WHOLE MEAL FLOUR D’ Egidio et al. New development for pasta products:immature wheat and functional components

6. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 MILKY PHASE total starch lysine FOS g protein content soluble sugars g necessity to use IWG flour in mixture with semolina in conventional pasta-making process CHARACTERISTICS OF IMMATURE WHEAT GRAINS (IWG) IWG: natural source of FOS Evolution of main components in durum wheat grains in comparison with MATURE WHEAT GRAINS, IWG present a: more balanced aminoacid composition (4-5 g lys /100 g protein) highercontent of soluble sugars (2-5%) higher content of total fiber (13-15%) lower level of starch (40-50%) and amylose (~20%) more relevant -amylase activity (2-15 IU/g d.m) similar protein content but higher level of albumins and globulins and lower/absence of gliadins and glutenins incapability of forming GLUTEN NETWORK D’ Egidio et al. New development for pasta products:immature wheat and functional components

7. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 AIM I characterization of protein and starch fractions of IWG in order to identify the more suitable technological processes to obtain FUNCTIONALFOODS • PROTEIN • Protein solubility • Accessibility of thiols • SDS-PAGE characterization • IMMUNOBLOTTING characterization • Surface hydrophobicity • STARCH • Total starch content • Amylose/Amilopectine ratio • Soluble Sugars • Viscoamylographic test • Microscopic investigation D’ Egidio et al. New development for pasta products:immature wheat and functional components

8. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 17th day 17th day • the decrease in albumins and globulins is associated to a noticeable increase in protein aggregates requiring urea and DTT for their solubilization • a marked decrease of both total and accessible thiol is assorbed at 17 daa insoluble proteins urea and DTT soluble proteins soluble proteins PROTEINS ACQUIRE A MORE COMPACT AGGREGATION STATE AFTER THE MILKY PHASE PROTEIN EVOLUTION DURING RIPENING (cv OFANTO) D’ Egidio et al. New development for pasta products:immature wheat and functional components

9. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 kDa 94 68 45 29 20 days after anthesis days after anthesis 9 13 17 21 28 9 13 17 21 28 GLIADIN GLIADIN SDS-PAGE IMMUNOBLOTTING characterization by SDS-PAGE and IMMUNOBLOTTING using commercial anti-gliadin antibodies confirmed that synthesis of the major gluten polypeptides begins at 17 days after anthesis, and that no immunoreactive gluten material was present at 9 or 13 daa IWG could be an interesting raw material for GLUTEN FREE FOOD D’ Egidio et al. New development for pasta products:immature wheat and functional components

10. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 STARCH PROPERTIES in IMMATURE andMATURE grains TEMPERATURE CONDITIONS MATURE GRAINS IMMATURE GRAINS MATURE GRAINS IMMATURE GRAINS Ø granules = 20-30 µm Ø granules = 2-3 µm Characteristics of starch can influence its digestibility IWG: potential ingredient for FUNCTIONAL FOOD D’ Egidio et al. New development for pasta products:immature wheat and functional components

11. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 BREAD FRESH PASTA AIM II study of the characteristics of IWG – enriched foods DRIED PASTA D’ Egidio et al. New development for pasta products:immature wheat and functional components

12. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 100% semolina 30% IWG 100% semolina 30% IWG +70% semolina 30% IWG +70% semolina COLOUR DETERMINATION furosine level= 755 mg /100g protein more than 50% LYSINE UNAVAILABLE HT LT 100% semolina LT HT EXPERIMENTAL PASTA-PRODUCTS PILOT PLANT SCALE (CRA-ISC, Rome) DRIED PASTA: SPAGHETTI SHAPE ( = 1.65mm) Formulation: • 100% semolina (REFERENCE SAMPLE) • 30% IWG and 70% semolina HT LT LOW TEMPERATURE: T = 50°C; 20 h HIGH TEMPERATURE: Tmax = 90°C; 8h D’ Egidio et al. New development for pasta products:immature wheat and functional components

13. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 dietetic pasta for diabetics (with INULIN) 1.63 59.0** 4.6 15.1 14.6** 4.5 Commercial 100% wholemeal semolina 6.9 2.38 3.6 63.5** 3.0 13.0** ** values reported on packaging CHEMICAL COMPOSITION OF DRIED PASTA* TOTAL STARCH SOLUBLE SUGARS RESISTENT STARCH FOS TOTAL FIBRE PROTEIN SPAGHETTI 3.1 0.70 74.9 1.7 11.1 1.1 100% semolina Experimental 30% IWG + 70% semolina 67.4 5.2 11.8 4.0 1.5 0.90 * data expressed as % db; all analyses in duplicate D’ Egidio et al. New development for pasta products:immature wheat and functional components

14. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 Cooking behaviour of pasta EVALUATION OF COOKING QUALITY according to D’Egidio et al., 1993 TOTAL SCORE * FIRMNESS OPTIMAL COOKING TIME (min) STICKINESS BULKINESS SAMPLE DISADVANTAGE OF DRIED PASTA 8.30 100% sem LT 61 73 55 55 8.00 63 60 70 60 30% IWG LT 11.30 69 73 65 68 DECREASE OF BENEFITS APPORTED BY ENRICHEMENT WITH IWG 100% sem HT 30% IWG HT 68 70 8.30 70 69 8.00 56 88 60 20 DIETETIC PASTA 65 * TOTAL SCORE 10 low quality –100 high quality WHOLEMEAL PASTA 65 70 60 8.30 before cooking Leaching of FOSafter cooking after cooking DISPITE THE LEACHING OF FOS INTO THE COOKING WATER, ENRICHED PASTA HAS A HIGHER FOS CONTENT THAN CORRESPONDING REFERENCE SAMPLE 30% IWG LT DIETETIC PASTA D’ Egidio et al. New development for pasta products:immature wheat and functional components

15. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 COOKED SPAGHETTI at optimal cooking time TIME OF ANALYSIS STEPS SIMULATION OF CHEWING portion of food containg 2g of available carbohydratesmixed with 5ml 20 mM sodium phosphate buffer (pH 6.9) containing 10 mm NaCl and 25 U of human salivary amylasegram of starch 5 min SIMULATION OF STOMACH DIGESTION acidification with HCl 8 M pH 2.0 “IN VITRO ” STARCH DIGESTION 1 h according to Brighenti et al., 1995 digestion at 37°C with hog pepsineper gram of starch SIMULATION OF SMALL INTESTINE DIGESTION correction of pH from 2.0 to 6.9 with NaOH 8 M mixture was placed in DIALYSIS BAGS with hogpancreatine 50 U per gram of starch and to simulate peristalsis a glass ball was inserted into the tube and used to mix the content of the tube every 15 min bags were suspended in separate sealed containers filled with 450 ml of phosphate buffer at 37°C 5 h every 30 min 1 ml sample of dialysate was taken for analysisof total dialysable reducing sugars by the 3,5-dinitrosalicylic acid method D’ Egidio et al. New development for pasta products:immature wheat and functional components

16. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 COMPARISON WITH COMMERCIAL PRODUCTS EFFECT OF IWG ADDITION PASTA for diabetics 100% semolina LT 100% semolina LT 30% IWG LT 30% IWG LT WHOLEMEAL PASTA Pasta enriched with IWG was characterized by a lower starch availability than the corrisponding 100% semolina Pasta enriched with IWG had a % of available starch lower than the product for diabetics GROWING AWARENESS FOR FOOD WITH LOW STARCH DIGESTIBILITY “IN VITRO STARCH DIGESTION” D’ Egidio et al. New development for pasta products:immature wheat and functional components

17. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 METHOD N° subjects = 10 (5 men; 5 women) 7 meals: Characteristics Mean ± sem Range • 4 spaghetti meals • 3 standard meals • (500 ml water + 50 g glucose) Age (years) 25,8 ± 0,5 22 - 28 Weight (kg) 66.4 ± 4.39 51 - 87 Height (m) 1.74 ± 0,03 1,60 - 1,87 0;15;30;45;60;90;120 min: finger prick blood samples (glucose determination: YSI 2000 STAT Glucose Analyser) BMI (kg/m2) 20,1 - 26,6 21,7 ± 0,8 Basal glycemia (mg/dl) 75,7 ± 8,8 63 - 90 Glycemix Index The Glycemic Index (GI) is defined as the incremental area under blood glucose response curve (AUC) elicited by a portion of food containing 50 g available carbohydrate expressed as the percent of AUC elicited by 50 g ofglucose taken by the same subjects. D’ Egidio et al. New development for pasta products:immature wheat and functional components

18. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 GLYCEMIC INDEX POST-PRANDIAL GLUCOSE RESPONSE The post-prandial glucose response was similar among pasta meals, showing a peak value at 30 min and a return to baseline values within 2 hours, with no significant differences in blood glucose concentration at any given time points. The calculated GI values of experimental pasta resulted slightly higher but not significantly different than those evaluated for commercial products. similar post-prandial blood glucose responses among pasta meals NO DIFFERENCES IN GLYCEMIC INDEX AMONG PRODUCTS D’ Egidio et al. New development for pasta products:immature wheat and functional components

19. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 CONCLUSIONS DRIED PASTA ENRICHED WITH IWG ACCEPTABLE COOKING QUALITY PRODUCT OF MEDIUM-LOW GLICEMIC INDEX HIGH FOS CONTENT BUT LEACHING OF THESE MOLECULES DURING COOKING WORK IN PROGRESS REDUCTION OF FOS LEACHING DURING COOKING FRESH PASTA ENRICHED WITH IWG (short cooking time) DRIED AND FRESH PASTA ENRICHED WITH PRE- COOKED IWG FLOUR REDUCTION OF STARCH DIGESTIBILITY PASTA WITH LOW GLYCEMIC INDEX D’ Egidio et al. New development for pasta products:immature wheat and functional components

20. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 Thank you! • Gabriella Bottega • Carolina Palandi • Chiara Balansino • Stefania Iametti • Franco Bonomi D’ Egidio et al. New development for pasta products:immature wheat and functional components

21. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 COOKED SPAGHETTI at optimal cooking time TIME OF ANALYSIS STEPS SIMULATION OF CHEWING portion of food containg 2g of available carbohydratesmixed with 5ml 20 mM sodium phosphate buffer (pH 6.9) containing 10 mm NaCl and 25 U of human salivary amylasegram of starch 5 min SIMULATION OF STOMACH DIGESTION acidification with HCl 8 M pH 2.0 1 h digestion at 37°C with hog pepsineper gram of starch “IN VITRO ” STARCH DIGESTION according to Brighenti et al., 1995 SIMULATION OF SMALL INTESTINE DIGESTION correction of pH from 2.0 to 6.9 with NaOH 8 M mixture was placed in DIALYSIS BAGS with hogpancreatine 50 U per gram of starch and to simulate peristalsis a glass ball was inserted into the tube and used to mix the content of the tube every 15 min bags were suspended in separate sealed containers filled with 450 ml of phosphate buffer at 37°C 5 h every 30 min 1 ml sample of dialysate was taken for analysisof total dialysable reducing sugars by the 3,5-dinitrosalicylic acid method The amount of released reducing sugars (consisting of dialysed fragments of digested starch plus the native reducing sugars) was expressed in maltose equivalent and as percentage of total available carbohydrates after adjustment for the rate of sugars diffusion RESULTS D’ Egidio et al. New development for pasta products:immature wheat and functional components

22. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 FOS DETERMINATION according to D’Egidio et al., 1993 PRELIMINARY EXTRACTION WITH ETHANOL 95% 1 h at 80°C FRUCTOSE CONTENT ENZYMATIC PROCEDURE (GOPOD) SOLUBLE SUGARS ethanolic extract residual GLUCOSE CONTENT WATER EXTRACTION CHEMICAL PROCEDURE (resorcine-HCl) 2 h at 105°C enzymatic attach FRUCTOSE CONTENT GLUCOSE CONTENT ENZYMATIC PROCEDURE (GOPOD) CHEMICAL PROCEDURE (resorcine-HCl) FRUCTAN D’ Egidio et al. New development for pasta products:immature wheat and functional components

23. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 SAMPLE FOS DETERMINATION EXTRACTION DISSOLUTION AOAC 1995 Revision March 1998 boiling water 10 min at 85°C sugar analysis 1 HYDROLYSIS amyloglucosidase 30 min at 60°C sugar analysis 2 HYDROLYSIS fructozyme 30 min at 60°C sugar analysis 3 Inulin= K (Glucose + Fructose) where K= conversion factorGlucose= from inulin = glucose from Hydrolisis II - Glucose from Hydrolisis I- Glucose in free sugar - glucose from disaccharides in free sugar (saccarose and maltose).Fructose= from inulin = fructose from Hydrolisis II - fructose in free sugar - fructose  from disaccharides in free sugar (saccarose) D’ Egidio et al. New development for pasta products:immature wheat and functional components

24. PASTA- PRODUCTS FRESH PASTA : TAGLIATELLE SHAPE Formulation: • 100% semolina (REFERENCE SAMPLE) • 30% IWG and 70% semolina DOUGH moisture = 30% LABORATORY PLANT SCALE sfoglia thickness = 2mm 2 CYCLES OF PASTEURIZATION 95°C 1 min before packaging 100°C 45 min after packaging 30% IWG +70% semolina 100% semolina D’ Egidio et al. New development for pasta products:immature wheat and functional components

25. III PASTA WORLD CONGRESS, 23rd – 26th October 2005 before cooking FUROSINE LEVEL after cooking 30 mg /100g protein 112 mg /100g protein the more porose the structure, the more available the starch Leaching of FOS after cooking “IN VITRO “STARCH DIGESTION 30% IWG 100% semolina 100% semolina LT 30% IWG LT 100% SEMOLINA 30% IWG HIGHER % OF DIGESTED STARCH THAN DRIED PASTA FRESH PASTA CHARACTERIZATION BROWN INDEX 100% semolina 30% IWG DRIED PASTA FRESH PASTA D’ Egidio et al. New development for pasta products:immature wheat and functional components

26. TOTAL PROTEIN